by strong adsorption behavioras the critical surface tension increases. The hydrophobic
`behavior of the surface is generally considered to occur whenthecritical surface tension is
`below 35 mN/m.Firstly, the reduction of the critical surface tension, the wetting of the
`surface, is associated with the hydrocarbonoil, ie the lipophilic behavior. The surface is
`considered both hydrophobic as well as resist oleophobic, wet with hydrocarbon oils so that
`the critical surface tension drops below 20 MN/meter. For example, cations on the silane
`surface can be used to generate a wide rangeofcritical surface tensions. Thus, the methods
`and compositions of the present invention include, for example, silane, 5, 6, 7, 8, 9, 10, 12,
`15, 20, 25, 30, 35, 40, 45, 50, 60, 70. , 80, 90, 100, 110, 115, less than 120 MN/M, or
`more, surface coatings may be used to achieve surface tension. Further, the methods and
`compositionsof the present invention are 115, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35,
`30, 25, 20, 15, 12, 10, 9, 8, 7., 6 MN/m,or less, surface coatings may be used, such as
`those containing silane, to achieve a surface tension greater than or equal to 6 MN/m.
`5175 Non-limiting examples of water contact angles and surface coatings include, for example,
`those containing silanes, such as those containing silanes, such as Arklesetal. (Silanes and
`Other Coupling Agents,Vol. in Surface Modification. 2009) Table 1 and Table 2, which are
`incorporated herein by reference in their entirety. The table is reproduced below.
`
`[0264]
`
`[0265]
`
`[0266]
`
`[0267]
`
`[0268]
`5194 The method of measuring the water contact angle can be any method knownin the art
`including static drop method, dynamic drop method, dynamic Wilhelmy method,single
`fiber Wilhelmy method, powder contact angle method,and thelike. Can be used.
`5197 In some cases, the surface of the substrate, as described herein in the present invention, or a
`portion of the surface of the substrate, as described herein, is non-curved, smooth, or
`planar. , On the equivalent of the relevant functionalized surface of the substrate to be
`hydrophobic or have a low surface energy, or about 90° ,95° , 100° , 105° ,110° ,
`115° , 120. It can be functionalized or modified to have a water contact angle measured
`greater than® , 125° , 130° ,135° ,140° ,145° or 150° .
`5203 The water contact angle of a functionalized surface described herein may refer to the contact
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`angle of a water drop on the functionalized surface in a non-curved, smooth or planar
`geometry.
`5206 In somecases, the surface of the substrate, as described herein in the present invention, or a
`portion of the surface of the substrate, as described herein, is non-curved, smooth, or
`planar. , On the equivalent of the relevant functionalized surface of the substrate to be
`hydrophilic or have high surface energy, or about 90° , 85° , 80° , 75° ,70° ,65° , 60
`Functionalized or modified to have a water contact angle measured greater than® ,55° ,
`50° , 45° , 40° , 35, 30° ,25° ,20° , 15° ,or 10° . obtain.
`5212 The surface of the substrate or a portion of the surface of the substrate may be
`functionalized or modified to be more hydrophilic or hydrophobic as comparedto the
`surface of the substrate or a portion of the surface of the substrate prior to
`functionalization or modification.
`
`[0269]
`5279 In somecases, the one or more surfaces are 90° , 85° , 80° ,75° ,70° ,65°
`as measured
`on one or more non-curved, smooth or planar equivalent surfaces. It can be modified to
`have a water contact angle difference of greater than” ,60° ,55° ,50° ,45° ,40° ,
`35° ,30° ,25° ,20° ,15° or 10° . In somecases, the surface of the microstructures,
`channels, sites of decomposition, defined reactor caps or other parts of the substrate are
`measured on equivalent surfaces of these structures that are not curved, smooth or planar.
`90° ,85° , 80° ,75° , 70° ,65° , 60° ,55° , 50° , 45° , 40° , 35° , 30° , 25° ,20° ,
`15° or It can be modified to have different hydrophobicities, corresponding to differences
`in water contact angles greater than 10° . Unless otherwise stated, the water contact angles
`described herein correspond to the measurementsobtained with the corresponding non-
`curved, smooth or planar equivalents of the surface.
`
`[0270]
`5233 Other methodsfor reactively grouping surfaces are described in US Pat. No. 6,028,189,
`which is incorporated herein by referencein its entirety. For example, hydrophilic degraded
`sites may be created byfirst applying or resisting protection over each degradedsite within
`the substrate. The unprotected area can then be coated with a hydrophobic agent to
`provide a non-reactive surface. For example, a hydrophobic coating is created by chemical
`vapor deposition of (tridecafluorotetrahydrooctyl)-triethoxysilane on the exposed oxide
`surrounding the protectedcircle. .. Finally, the protective agent, or resist, can be removed to
`exposeareas of the substrate location for further modification and oligonucleotide
`synthesis. In some embodiments,the initial modification of such unprotected regions can
`resist further modifications, preserving their surface function, while the newly unprotected
`regions are subsequently modified. Can be exposedto.
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`[0271]
`5247 Multiple Parallel Microfluidic Reactions In another aspect, a set of systems and methodsfor
`performing parallel reactions are described herein. The system may include two or more
`substrates that may be hermetically sealed, eg, releasably hermetically sealed to each other,
`to form a reaction volume or multiple reactors that are individually addressable upon
`closure. A newset of reactors can be formed by releasing thefirst substrate from another
`substrate and aligning it with the third substrate. Each substrate can carry reagents(eg,
`oligonucleotides, enzymes, buffers, solvents) for the desired reaction. In some embodiments,
`the system includesa first surface of a plurality of decomposedlocationsata first suitable
`density and a plurality of decomposed reactor caps at a second suitable density. The system
`may align a plurality of disassembled reactor caps at a disassembled location on the first
`surface that form a temporary seal betweenthefirst surface and the cap element. The
`temporary seal betweenthe aligned substrates is about at least about
`2,3,4,5,6,7,8,9,10,11,12,13,14,15 at a location on thefirst surface. 16, 17, 18, 19, 20, 25,
`30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200 or those It may
`be physically divided into groupsof less than or more places. The set of parallel reactions
`described herein can be carried out by the methods and compositions of the present
`invention. A first surface with a plurality of decomposed locationsat a first suitable density
`and a cap elementwith a plurality of decomposed reactor caps at a second suitable density
`can bealigned; The system aligns a plurality of resolved reactor caps in disassembled
`locations onthefirst surface that form a temporary seal between the first surface and the
`cap element, thereby causing The location is about at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,
`11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35. , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
`90, 95, 100, 125, 150, 200 orless, or more, physically Can be divided. A first reaction is
`performed to formafirst set of reagents. The cap element can be released from the first
`surface. Upon release, each reactor cap mayretain at least a portion ofthefirst set of
`reagents in the previously sealed reaction volume.
`5273 The plurality of decomposed places are about,at least about 1, about 2, about 3, about 4,
`about5, about 6, about 7, about 8, about 9, about 10, about 15, about 1 mm<2>. 20, about
`
`25, about 30, about 35, about 40, about 50, about 75, about 100, about 200, about 300,
`
`about 400, about 500, about 600, about 700, about 800, about 900, about 1000, About
`
`1500, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about
`
`8000, about 9000, about 10,000, about 20,000, about 40,000, about 60,000, about
`80,000, about 100,000, or about 500,000,Alternatively, it may be less than them. In some
`embodiments,the plurality of resolved locations can have a density of about at least about
`100, or less per mm<2>. In some embodiments, the plurality of disassembled reactor caps
`can have a density of about, at least about 1, or less per mm<2>. In some embodiments, the
`plurality of reactor caps are about, at least about 1, about 2, about 3, about 4, about5,
`about6, about 7, about 8, about 9, about 10 per mm<2>., About 15, about 20, about 25,
`about 30, about 35, about 40, about 50, about 75, about 100, about 200, about 300, about
`
`400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500,
`
`about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000,
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`

`about 9000, about 10,000, about 20000, about 40,000, about 60,000, about 80,000, about
`100,000. , Or about 500,000,or less. The method described herein comprises providing a
`second surface with a plurality of decomposedsites at a third density, a plurality of
`decomposedsites with a plurality of decomposedsites on the second surface. Aligning the
`reactor cap and forming a seal, typically temporary or releasable, between the second
`surface and the cap element. The newly formed seal has a location on the second surface of
`aboutat least about 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16., 17, 18, 19, 20, 25, 30, 35, 40,
`45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200 or less May bephysically
`divided into groupsof, or more locations than.
`5297 The second reaction may optionally be carried out using a portion ofthefirst set of reagents,
`thereby forming a second set of reagents. The cap element can be released from the second
`surface. Upon release, each reactor cap mayretain at least a portion of the second set of
`reagents in the previously sealed second reaction volume. In some embodiments, the
`second surface with the plurality of disassembled locations has at least about 1, about 2,
`about3, about 4, about 5, about 6, about 7, about 1 mm<2>. 8, about 9, about 10, about 15,
`
`about 20, about 25, about 30, about 35, about 40, about 50, about 75, about 100, about
`
`200, about 300, about 300, about 400, about 500, About 600, about 700, about 800, about
`
`900, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about
`
`6000, about 7000, about 8000, about 9000, about 10000, about 20000, aboutIt can be
`
`40,000, about 60,000, about 80,000, about 100,000, or about 500,000,or less. Various
`systems, methods and instrument uses of embodiments are described herein.
`
`[0272]
`5312 The system assembly can include any number of dynamic wafers and any numberofstatic
`wafers. For example, the system can include three substrates in rows and four substrates in
`columns. The transport system may include three static wafers (or substrates) and one
`dynamic wafer (or substrate). Dynamic wafers can be moved or transported between
`multiple static wafers. Dynamic wafers can be transported betweenthreestatically mounted
`wafers. In some embodiments, the dynamic wafer can have a diameter of about 50, 100,
`150, 200 or 250 mm,or2, 4, 6 or 8 times higher. The dynamic wafer can be attached to a
`temperature controlled vacuum chuck. The system of the present invention allows for a
`configuration in which a dynamic wafer can movein the Z direction, which can be about
`0.01, 0.05, 0.1, 0.5, 1, 1.5, 2 or With control of the z-position of 3 4m or less, it can be in a
`direction perpendicular to the surface of the wafer facing the second wafer surface, and
`theta_z of the wafer, between the normals of the surfaces of the two wafers facing each
`other, Can be matched,for example, by matching another pattern on the static wafer with a
`pattern on the dynamic wafer within a tolerance. The wafer positioning tolerance is about
`5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 in termsof the
`difference in the rotation angle of the xy plane. It can be 400, 450 or 500 microradians, or
`less. Wafer positioning tolerances may be about 50 microradiansorless in the difference in
`rotation angles in the xy plane. The wafer positioning tolerance is about 0.01, 0.05, 0.1, 0.5,
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`

`1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 in the x-direction distance. , 12, 13, 14 or 15 “morless. The
`waferpositioning tolerance is about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 in the
`y-direction distance. , 12, 13, 14 of 15 “morless. Wafer positioning tolerances may be
`about1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 microradians or less at rotation angles in the xy plane in
`the z-direction. In some embodiments, the wafer positioning tolerance can be about 5
`microradiansor less at an angle of rotation of the xy planein the z-direction.
`5336 IN some embodiments, the wafer positioning tolerance is about 0.01, 0.05, 0.1, 0.5, 1, 1.5,2,
`2.5, 3, 3, in the z-direction distance. It can be 3.5, 4, 4.5 or 5 4m orless. In some
`embodiments, the wafer positioning tolerance can be a distancein the z-direction of about
`0.5 “£m,orless.
`
`[0273]
`5343 In some cases, systems and methodsfor performing a set of parallel reactions include third,
`fourth, with cap elements with multiple decomposed sites, and/or multiple decomposed
`reactor caps. Further includesa fifth, sixth, seventh, eighth, ninth or tenth surface. The
`third, fourth,fifth, sixth, seventh, eighth, ninth or tenth surface can be lined up to form a
`temporary seal and corresponding cap element between the two surfaces. , Which allows to
`physically partition the location on the surface and/orthe reactor. Thethird, fourth,fifth,
`sixth, seventh, eighth, ninth or tenth reaction is the previous reaction(i.e. the second,third,
`fourth, fifth, sixth, sixth of the reagents). 7th, 8th, 9th set of reactions) can be carried out
`using a portion of the reagents retained thereby, whereby the 3rd, 4th, 5th, 6th, 7th, 8th,
`8th Form the ninth or tenth reagent. Each of the cap elements described herein can be
`released from its corresponding surface, where the reactor cap can hold at least a portion of
`the previous set of reagents in another reaction volume. In somecases, the second surface
`with a plurality of resolved locations may have a density of at least 2/mm<2>. In some
`embodiments,the plurality of decomposed locations is about, at least about 1, about 2,
`about3, about 4, about 5, about 6, about 7, about 8, about 9 per mm<2>., About 10, about
`15, about 20, about 25, about 30, about 35, about 40, about 50, about 75, about 100,
`
`about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900,
`
`about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about 6000,
`
`about 7000, about 8000, about 9000, about 10000, about 20000, about 40,000, about
`60,000, about 80,000 , About 100,000, or about 500,000,or less. The portion of the
`reagent retained each time can be different and can be controlled to be the desired portion,
`depending on the reaction being performed.
`
`[0274]
`5368 The presentinvention, in various embodiments, contemplates a system for performing a set
`of parallel reaction comprising a first surface with a plurality of disassembled locations and
`a plurality of disassembled reactor caps. As described in further detail elsewhere herein, a
`cap element comprising a plurality of disassembled locations and a plurality of solution
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`

`reactor caps are combined to form a plurality of disassembled reactors. In some cases, the
`degradedlocation of the first surface of the first substrate may include a coating of
`reagents. The decomposedlocation of the second surface of the second substrate may
`include a coating of reagents. In some embodiments, the reagent coating can be covalently
`linked to the first or second surface. In some cases, when thereis a third, fourth, fifth, sixth,
`seventh, eighth, ninth or tenth surface, each surface may comprise a respective coating of
`reagent.
`
`[0275]
`5382 The coating of reagents onthefirst surface or the second surface can include
`oligonucleotides. The oligonucleotides are of any length, eg, at least 25, 50, 75, 100, 125,
`150, 175, 200, 225, 250, 275, 300 bpor longer, as described further herein. possible.
`Upon sealing the disassembled location with the reactor cap, the oligonucleotide contained
`within the coating of reagents can be released. Various reactions, such as oligonucleotide
`amplification reactions, PCA, sequencing library generation, or error correction can be
`performed within multiple disassembled reactors.
`
`[0276]
`5392 Oligonucleotides may be preparedbya variety of suitable methods,as described in further
`detail elsewhere herein, and by any suitable method as knownin the art, such as by
`enzymatic cleavage well knownin the art. It can be released from the coated surface. Other
`methods of cleavage knownin the art include restriction enzymes such as Mlyl, or other
`enzymes, or single-stranded or double-stranded, such as, but not limited to, uracil DNA,
`glycosylase (UDG) and DNA endonucleaseIV. Including the use of a combination of
`enzymescapable of cleaving double-stranded DNA. Other cleavage methods knownin the
`art also include, but are not limited to, chemical (base-labile) cleavage of DNA molecules or
`optical (due to photo-labile) cleavage from the surface of the present invention. Can be used
`advantageously. PCR or other amplification reactions, while they are tethered to the
`substrate, can also be utilized to produce the building material for gene synthesis by
`copying oligonucleotides. Methods of releasing oligonucleotides are described in
`W02007137242 and US Pat. No. 5,750,672, which are hereby incorporated by reference
`in their entirety.
`
`[0277]
`5409 In somecases, releasing the cap elementfrom thefirst surface and releasing the cap element
`from the second surface can occur at different rates. The amountof the portion of the
`reagentthat is retained in releasing the cap element from the corresponding surface can be
`controlled by the surface or the surface energy of the capping element and the
`corresponding surface. In some cases, the first or second surface comprises different
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`

`surface tensions, surface energies, or hydrophobicities with a given liquid such as water. In
`some embodiments, the degraded location of the first surface comprises high surface
`energy, surface tension or hydrophobicity. The difference in surface energy, or
`hydrophobicity, of the capping element and the corresponding surface can be a parameter
`for controlling the portion of the reagent retained on release. The first volume and the
`second reaction can bedifferent.
`
`[0278]
`5423 In somecases, the pressure outside the cracked reactor may be greater than the pressure
`inside the cracked reactor. In other cases, the atmospheric pressure outside the cracked
`reactor may beless than the pressure inside the cracked reactor. The pressure difference
`(or pressure difference) inside the cracked reactor and outside the cracked reactor can
`affect the temporary sealing of the cracked reactor. Due to the modification of the surface
`energy or hydrophobicity of the first surface and the second surface, the pressure
`difference causes a curvedorlinear air/liquid flow in the gap between the first surface and
`the reactor cap of the second surface. Interfaces can result. Furthermore, the force required
`to release the cap element from the surface can be controlled by the pressure differential
`and the different surface energies. In some cases, the surface can be modified to have
`different surface energies and pressure differences so that the capping element can be
`easily released from the surface.
`
`[0279]
`5438 The first or second reaction, or any reaction after the second reaction, can be assayed for a
`variety of molecules, such as any suitable reaction described herein or Knownin theart.
`Alternatively, it may include a biochemical assay. In some embodiments, the first or second
`reaction can include a polymerase cycling assembly. In some embodiments,thefirst or
`second reaction is enzymatic gene synthesis, annealing and ligation reactions, simultaneous
`synthesis of two genesvia hybrid genes, shotgun ligation and coligation, insert gene
`synthesis, DNA synthesis. It may include gene synthesis via single strand, template-directed
`ligation, ligase chain reaction, microarray mediated gene synthesis, solid phase assembly,
`Sloning building block technology, or RNA ligation mediated gene synthesis. A reaction or
`method of conducting a set of parallel reactions involves cooling the cap element or cooling
`the first surface (second surface), which may facilitate.
`
`[0280]
`5452 A general process workflow of the methods and compositions of the present invention using
`the system described herein is illustrated in FIG.
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`

`[0281]
`5457 Use of Auxiliary Equipment In one aspect, the present invention relates to systems and
`methodsfor oligonucleotide synthesis.
`5459 Systemsfor the synthesis of oligonucleotides may include a scanning deposition system.
`Systems for oligonucleotide synthesis typically include a plurality of printheads, a
`functionalized surface, and a plurality of disassembled locations anda first substrate having
`an inkjet printer (eg, oligonucleotides). Synthetic wafers). Each printheadis typically
`configured to place one of the various building blocks for the reaction to be carried out at
`the resolvedsite of the first substrate (eg, nucleotide building block for phosphoramidite
`synthesis). It The degraded location of the oligonucleotide synthesis wafer can be in a
`microchannel as described in further detail herein. The substrate is provided in the flow cell
`by providing a continuousflow, such as one containing the necessary reagents (eg, oxidant
`in toluene) or solvent(eg, acetonitrile) for the reaction in the decomposed location. Can be
`sealed with, which allows for precise control of dose and concentration at the site of
`synthesis (eg, the degraded position of the oligonucleotide synthesis wafer). A flow of an
`inert gas such as nitrogen can be used to dry the substrate by the enhanced evaporation of
`a typically volatile substrate. Various means, such as vacuum source/vacuum pumpsor
`vacuum tanks, provide a reduced phasedrelative pressure (negative pressure) or vacuum to
`improve drying and reduce residual water content and any droplets on the surface. Can be
`used to generate. Therefore, the pressure immediately surrounding the substrate orits
`decomposedlocation is about 100, 75, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.1. , 0.05,
`0.01 mTorr,or less.
`
`[0282]
`5481 FIG. 3 shows an example of a system for oligonucleotide synthesis. Thus, oligonucleotide
`synthesis wafers are configured to provide, via an inlet manifold, and optionally an outlet
`manifold, a degraded location for oligonucleotide synthesis with the required bulk reagents.
`Large amounts of reagents include any suitable oxidant, reagent, support, such as de-block,
`acetonitrile or nitrogen gas, for oligonucleotide synthesis that is generally needed among
`multiple degraded sites in various embodiments., A solvent, a buffer or a gas. The
`printhead of the inkjet printer can movein the XY directions with respect to the
`manageable position of the first substrate. A second substrate, such as a cap element, can
`be movedin the Z direction, and optionally in the X and Y directions, as described in more
`detail elsewhere herein. Forming a plurality of disassembled reactors for sealing with the
`substrate. Alternatively, the second substrate may be fixed. In such cases, the synthetic
`substrate may movein the Z direction, optionally sealing the second substrate side by side
`in the X and Y directions. The synthesized oligonucleotide can be delivered from thefirst
`substrate to the second substrate. An appropriate amountof fluid flows through the suction
`manifold of the first substrate and the decomposedsite to the second substrate to deliver
`the reagentfrom thefirst substrate/the decomposedsite to the second substrate. Facilitate.
`In another aspect, the present invention is directed to a system for assembly of
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`

`oligonucleotides including wafer handling.
`
`[0283]
`5502 In various embodiments, the present invention utilizes a system for scanning deposition.
`Scanning deposition systems can include inkjets that can be used to deposit reagents for
`disassembled locations or microwells. In some embodiments, the scanning deposition
`system can use organic solvents or inks. In some cases, the scanning deposition system can
`include multiple silicon wafers, typically about 200 mm in diameter. In some cases, the
`entire system may be located and functional in a pressure controlled enclosure. The
`scanning deposition system may include a working envelope, a printhead assembly, a flow
`cell assembly, and/or a service envelope. In some cases, the printhead assembly can move,
`but the flow cell assembly remains stationary. A scanning deposition system may deposit
`one or more substrates/wafers, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more
`
`substrates/wafers. One or more flow cells can be retained, such as 2, 3, 4, 5, 6, 7, 8, 9, 10,
`15, 20, 30, 40, 50, or more flow cells to be maintained. The wafer may remain fixed in the
`flow cell. In some cases, the system mayfacilitate substrate alignment through theta_z
`automation. The work envelope may include a region containing a scanning direction travel,
`eg, about (n-1) printhead pitch+wafer diameter=9*20 mm=380 mm... A suitable actuation
`envelope can be foreseen with an equivalent configuration. The service envelope may
`include a printhead placed for maintenance. In some cases, the service envelope may be
`environmentally isolated from the larger box. In various embodiments, systems for the
`methods and compositions described herein include scanning deposition systems for
`oligonucleotide synthesis, oligonucleotide assembly, or more generally reagent generation.
`
`[0284]
`5525 The plurality of disassembled locations and the plurality of disassembled reactor caps may
`be arranged on a microstructure having interconnectivity or fluidic communications. Such
`fluid communication allows for washing and perfusion of new reagents, either as droplets
`or using continuousflow for different steps of the reaction. The fluid communication
`microchannels mayinclude inlets and outlets to and/or from a plurality of disassembled
`locations. The inlet and/or outlet can be made by any method knownin theart. For
`example, inlets and/or outlets can be provided on the front side and behind the substrate. A
`method of making inlets and/or outlets is described in US Patent No. 20080308884A1,
`which is incorporated herein by referencein its entirety, to provide suitable
`microstructured components byfront side lithographic and etching processes. Making;
`drilling holes from the back side of the substrate with precise alignment with front side
`microstructuresto provide inlets and/or outlets to and/or from the micromechanical
`structure The inlets and/or outlets, which may include, are fluids flowing through a thin
`gap supplied by a manifold and may be Hele-Shaw typeflow cells. As shownin FIG. 9A, the
`substrate described herein may form part of a flow cell. The flow cell can be closed by
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`

`sliding the lid over the top of the substrate (ie, the wafer) and tightened into position to
`form a pressure seal around the edge of the substrate. In some embodiments, the
`hermeticity may be sufficient to seal against a vacuum or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
`atmospheres. Reagents can be plugged into a thin gap beneath the substrate (ie, wafer) and
`flow through the substrate. The reagents can then be collected with a tapered waste
`collector, as illustrated in Figure 9B. In some embodiments,after the final solvent cleaning
`step, the wafer can be drained, eg, passed through the bottom of the assembly and then
`purged with nitrogen. Thereafter, the chamber is 50%, 30%, 30%, 20%, 10%, 9%, 8%, 7%,
`6%, 5%, 4%, 3%, 2%, 1%, 0% by volume. Vacuum to dry residual solvent in any
`microstructure that reduces residual liquids and moisture to 1%, 0.01%, 0.001%, 0.0001 %,
`0.00001% or less. Can be pulled downto.
`5551 The chamberis thenless than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 200, 300,
`400 or 1000 mTorr. As can be seen, a vacuum can be pulled down to reduce the pressure
`around the substrate. In some cases, the chambercan befilled with nitrogen after the
`vacuum step and the roof can be reopenedandslid to allow accessto ancillary components
`of the system (eg printer). can be slide open). In somecases,the flow cell can be opened. As
`illustrated in FIG. 9B, the substrate/wafer may be loaded with disposed sideways waste
`manifolds. This arrangement mayallow easier inkjet access to the wafer. At this point, the
`reagents can be deposited in the microwells. In some embodiments, the lid of the
`disassembled housing(ie, flow cell) can function as a waste collector, and reagentliquid can
`flow there. The arrowsin Figures 9B and 9Crepresenttypical flow directions for reagents.
`In some cases, reagents can pass through holes in the substrate(eg, a silicon wafer) and
`enter through a thin gapin the bottom andbe collectedin a waste collector,as shownin
`FIG. 9C. In some examples, the gas may be purged via an upperor lower manifold, eg,
`through the bottom or top of the flow cell, to exclude liquid. The outlet or inlet port can be
`connected to a vacuum until completely dry. As illustrated in FIG. 10, the vacuum port can
`be connected to the unwantedside orthe inlet side. In some implementations, there can be
`multiple pressure reliefholes through the substrate (ie, wafer). The plurality ofholes may
`be about 1000, 5000, 10,000, 50,000, 100,000, 500,000 or 2,000,000 or more. In some
`cases, the plurality of holes may be 5 million or more. In some cases, the microstructure for
`synthesis functions as a pressurerelief hole, as described in further detail elsewhere herein.
`These holes can allow gas to pass from oneside of the wafer so that the disassembled
`housing is emptied to dry the substrate.
`5573 In some cases, for example, whenair is driven from the waste collector side, the waste
`collector side air pressure (Pwaste) may be maintained at substantially the same level as the
`inlet side air pressure (Pinlet). In some embodiments, a port connecting the inlet manifold
`to the waste collector may be used. Thus, someof the steps described herein, such as
`scanning,flooding, cleaning, removal and/or drying, can be performed without shipping the
`wafer substrate.
`
`[0285]
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`21-07-2020
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`133
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`

`

`5582 The decomposed reactor, which is formed by sealing the first and second substrates,is
`surrounded by a chamberby controlled humidity, air volume, vapor pressure and/or
`pneumatic molding andis controlled by a controlled environment. An assembly may be
`formed. In some embodiments, the humidity of the chambercan be saturated or can
`eliminate about 100% evaporation of liquid from the decomposedreactor during the
`reaction. For example, in some embodiments, the humidity is about at least about 100%,
`99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%. , 95.5%, 95%, 94%, 93%, 92%, 91%, 9O%,
`
`89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% , 80%, 75%, 70%, 65%, 60%, 55%, 50%,
`
`45%, 40%, 35%, 30% or 25% or more.
`
`[0286]
`5594 The systems described herein, such as those with the controlled environment assembly
`described above, may include a vacuum device/chuck and/or temperature control system
`operably connected to multiple reactors. The substrate can be placed on a vacuum chuck.
`The vacuum chuck mayinclude surface irregularities located beneath the substrate. In
`various embodiments,the surface irregularities can include channels or recesses. The
`vacuum chuck maybein fluid communication with the substrate for drawing gas from the
`space defined by the channels. Methodsof maintaining a substrate on a vacuum device are
`described in further detail in US Pat. No. 8,247,221, which is incorporated herein by
`referencein its entirety.
`
`[0287]
`5606 In various embodiments, the substrate (eg, a silicon wafer) can be located on a chuck, such as
`the vacuum chuck described above. FIG. 10 illustrates a single groove vacuum chuck
`system assembly and a piece of sintered metal between the substrate and the temperature
`controller. The vacuum chuck can include a single groove with appropriate dimensions for
`holding the substrate. In some embodiments, the vacuum chuckis designed so that the
`substrate can be held in place during one or more of the methods described herein. The
`vacuum chuckillustrated in FIG. 10A by way of example includes a single 1-5 mm groove
`approxi